Complete DNA sequence of Yersinia enterocolitica serotype 0:8 low-calcium-response plasmid reveals a new virulence plasmid-associated replicon

Abstract

The complete nucleotide sequence and organization of the Yersinia enterocolitica serotype 0:8 low-calcium-response (LCR) plasmid, pYVe8081, were determined. The 67,720-bp plasmid encoded all the genes known to be part of the LCR stimulon except for ylpA. Eight of 13 intact open reading frames of unknown function identified in pYVe8081 had homologues in Yersinia pestis plasmid pCD1 or in Y. enterocolitica serotype 0:9 plasmid pYVe227. A region of approximately 17 kbp showed no DNA identity to pCD1 or pYVe227 and contained six potential new genes, a possible new replicon, and two intact insertion sequence (IS) elements. One intact IS element, ISYen1, was a new IS belonging to the IS256 family. Several vestigial IS elements appeared different from the IS distribution seen in the other LCR plasmids. The RepA proteins encoded by Y. enterocolitica serotype 0:8 pYVeWA and pYVe8081 were identical. The putative pYVe8081 replicon showed significant homology to the IncL/M replicon of pMU407.1 but was only distantly related to the replicons of pCD1 and pYVe227. In contrast, the putative partitioning genes of pYVe8081 showed 97% DNA identity to the spy/sopABC loci of pCD1 and pYVe227. Sequence analysis suggests that Yersinia LCR plasmids are from a common ancestor but that Y. enterocolitica serotype 0:8 plasmid replicons may have evolved independently via cointegrate formation following a transposition event. The change in replicon structure is predicted to change the incompatibility properties of Y. enterocolitica serotype 0:8 plasmids from those of Y. enterocolitica serotype 0:9 and Y. pestis LCR plasmids.

FIG. 1

Map of pYVe8081 showing significant genes, IS elements, and replication and partition regions. The direction of transcription is clockwise for genes shown inside the circle and counterclockwise for genes shown outside the circle. Green boxes indicate genes comprising the LCR stimulon, and a purple box indicates yadA. Yellow boxes indicate genes with replication and partition functions. Pink boxes indicate previously identified genes of unknown function, and potential new genes are indicated by light blue boxes. IS elements are indicated by dark blue boxes. IS remnants shown in this figure are discussed in the text. IS-like indicates IS element remnants with less than 90% DNA identity to the GenBank match. ISYen-p is a truncated homologue of ISYen1. The positions of the virB operon (yscN to yscU) and the virC operon (yscA to yscM1) are noted in boldface on the outside of the circle. The inner circle shows the scale in kilobase pairs.

FIG. 2

Comparison of LCR plasmid maps of pYVe8081, pYVe227, and pCD1. Only representative genes are shown, to facilitate orientation. The maps of pYVe227 (21) and pCD1 (39) are redrawn. Arrows outside the circles indicate the direction of transcription. YadA′ indicates the yadA pseudogene in pCD1, and Tn2502 is the arsenic resistance transposon in pYVe227. Regions in pCD1 involved in the inversions referred to in the text are shown in gray and numbered. The dots represent GTATT direct repeats in pYVe8081, pYVe227, and pCD1.

FIG. 3

Comparison of the sycT-yopD region in pYVe8081 to corresponding regions in pYVe227 and pCD1. The different colors represent different DNA segments. White segments are not present in pYVe8081. Arrows under the plasmid maps show the orientation of DNA segments. Numbers indicate nucleotide positions in base pairs for each plasmid. Sequences of pYVe227 and pCD1 are from the GenBank database (accession no. AF102990 and AF074612, respectively). Remnants orf61*, orf60*, and orf54* are truncated ORFs in pYVe8081 that show homology to intact genes (orf61, orf60, and orf54) in pCD1. Genes encoding YopM and ORF91B are also labeled. Arrows within genes show the direction of transcription. White arrows indicate the positions of the long repeated sequences R1, R2, and R3, referred to in the text. Only intact IS elements are shown.

FIG. 4

Nucleotide sequence of the putative replication region in pYVe8081. The numbers correspond to base pair positions in pYVe8081. The sequence shown is single stranded, with the deduced amino acid sequences given in single-letter amino acid code. The deduced amino acid sequence of RepB is shown above its nucleotide sequence. Possible start codons are shown in boldface, and an arrowhead following the gene name indicates the direction of transcription. An asterisk in the amino acid sequence depicts a nonsense codon. Possible Shine-Dalgarno sequences have a single line underneath them. Dots depict nucleotides that are identical in pYVe8081 and the pMU407 replicon. The possible −10 and −35 sequences for the antisense RNA (RNAI) are boxed. RNAI is underlined in boldface with an arrow that indicates the direction of transcription. Facing arrows indicate possible stem-loop structures in RNAI and repB. The double line under bp 1 to 9 denotes a possible DnaA protein-binding site. Arrows labeled R1, R2, and R3 indicate direct repeats within the AT-rich region.

FIG. 5

Comparison of the sequences of the stem-loop structures predicted for RNAI molecules of pYVe8081, IncL/M (2), and IncB (49) plasmids based on the computer program of Zuker (27, 62). The conserved loop sequence (CGCCAA) and the GC-rich region in the major stem-loop are shown in boldface.

FIG. 6

Unrooted phylogenetic tree of the translated products of repA genes from the extended RepFIIA family. Alignments were generated using the CLUSTAL program (DNAStar). Incompatibility groups are shown in parentheses. The replication initiator protein from rolling-circle replicating plasmid pT181 (31, 32) is included as an unrelated protein for a control. Buchnera aphidicola plasmids (50, 58) and their RepA (GenBank) accession numbers are as follows: pBPp1, CAA07300; pBTs1, CAA72701; and pBTc1, CAA72709. Other plasmids and their RepA (GenBank) accession numbers are as follows: pCD1, AAC69762; pYVe227, AF102990; pNR1, CAA26168; pSU316, AAA98204; pCol1b-P9, AAA23191; pMU707, AAA98176; pMU407.1, AAA87028; pR387, AAA98310; pYVe8081, AY026194; pYVeWA, AY026195; and p307, AAB17112.